Assessment of virulence factors and antimicrobial resistance among the Pseudomonas aeruginosa strains isolated from animal meat and carcass samples

Abstract Background Pseudomonas aeruginosa bacteria are emerging causes of food spoilage and foodborne diseases. Raw meat of animal species may consider a reservoir of P. aeruginosa strains. Objectives The present survey was done to assess the prevalence, antibiotic resistance properties and distribution of virulence factors among the P. aeruginosa strains isolated from raw meat and carcass surface swab samples of animal species. Methods Five hundred and fifty raw meat and carcass surface swab samples were collected from cattle and sheep species referred to as slaughterhouses. P. aeruginosa bacteria were identified using culture and biochemical tests. The pattern of antibiotic resistance was determined by disk diffusion. The distribution of virulence and antibiotic resistance genes was determined using polymerase chain reaction. Results Forty‐seven of 550 (8.54%) examined samples were contaminated with P. aeruginosa. The prevalence of P. aeruginosa in raw meat and carcass surface swab samples were 6.57 and 12%, respectively. P. aeruginosa isolates showed the maximum resistance rate toward penicillin (87.23%), ampicillin (85.10%), tetracycline (85.10%), gentamicin (65.95%) and trimethoprim (57.44%). The most commonly detected antibiotic resistance genes were BlaCTX‐M (53.19%), blaDHA (42.55%) and blaTEM (27.65%). The most commonly detected virulence factors was ExoS (42.55%), algD (31.91%), lasA (31.91%), plcH (31.91%) and exoU (25.53%). Conclusions Meat and carcass surface swab samples may be sources of resistant and virulent P. aeruginosa, which pose a hygienic threat in their consumption. However, further investigations are required to identify additional epidemiological features of P. aeruginosa in meat and carcass surface samples.


INTRODUCTION
Raw meat of animal species may act as a reservoir of several foodborne pathogens, responsible for both food spoilage and also the occurrence of foodborne diseases (Heredia & García, 2018). Pseudomonas aeruginosa (P. aeruginosa) is a food microorganism and opportunistic human pathogen extensively distributed in food and the environment (Gu et al., 2016). It is usually present in environmental sources, including soil and water. It is supposed that it can be found on vegetables, fruits and meat. Meat storage under aerobic conditions allows P. aeruginosa growth and proliferation even in different temperatures (Neto et al., 2012). It can easily develop in milk, fish, meat and dairy samples stored aerobically at low temperature. The growth of the bacterium is responsible for off-flavours, pigmentation, slime and malodour production in meat and derived products (Stellato et al., 2017). Previous studies showed that drinking water contaminated with P. aeruginosa may cause foodborne infections (Wei et al., 2020;Vukić Lušić et al., 2021). Additionally, there is growing evidence implication of the P. aeruginosa in foodborne infections (Bricha et al., 2009;Nawaz & Bhattarai, 2015;Virupakshaiah & Hemalata, 2016). However, there is no available data about the occurrence of foodborne diseases after the consumption of meat contaminated with P. aeruginosa. Thus, there is a crucial need to determine the role of raw meat of animal species as a reservoir of P. aeruginosa.
P. aeruginosa contained several types of virulence factors responsible for the pathogenesis of diseases and related infections. Phenazine operons (phzH, phzM and phzS) secrete the precursor proteins to encode three phenazine compounds responsible for the intracellular oxidative effects (Heggins et al., 2018). Elastase gene A and B (lasA and lasB), exoenzymes (exoS, exoT, exoU and exoY), haemolytic and non-haemolytic Phospholipase C (plcH and plcN) and alginate-encoded genes (algD and algU) are other essential virulence factors of the P.
Recent reports revealed the high resistance rate of P. aeruginosa strains toward different types of antimicrobial agents (Pang et al., 2019;Soares et al., 2020). Antibiotic-resistant P. aeruginosa strains caused more severe infections for a longer time with a higher economic burden (Meliani 2020 Abdelrahman et al., 2020).
According to the uncertain role of meat and animal carcasses as sources of P. aeruginosa transmission to the human population and also a risk of foodborne diseases, the present study was done to assess the prevalence and antibiotic resistance rate and distribution of virulence factors and antibiotic resistance genes of P. aeruginosa strain isolated from raw meat and carcasses surface swab samples of bovine and ovine species. Figure 1 shows the map of the study area.

Samples
The sample size was determined using the following formula. A total of 550 samples, including raw cattle (n = 175) and sheep (n = 175) meat and cattle (n = 100) and sheep (n = 100) carcasses surface swab samples were randomly collected from animals referred to slaughterhouses in North Iran. Raw meat samples (100 g) were collected from the tight muscle using sterile plastic bags. Carcass swab samples were taken from a 20 cm 2 area of the tight muscle after bleeding, skinning, eviscerating and washing stages using the swabbing technique. Swab samples were transferred using sterile tubes containing 0.1% peptone water solution. Samples were transferred in refrigerated containers at 4 • C. Samples transportation and processing were done within 2 h after collection.
where p is the H. pylori mean prevalence in recent studies, Z is the abscissa of the standard curve that cuts off an area α at the tails (1.96), d is the acceptable sampling error and N is the sample size.

Antimicrobial resistance analysis
To investigate the pattern of antibiotic resistance of P. aeruginosa isolates, the simple disk diffusion method (Kirby Baeur) was

Assessment of antibiotic resistance genes and virulence factors
Besides, UVI doc gel documentation systems (Grade GB004; Jencons PLC, London, UK) were used to analyse images (Ranjbar et al., 2019). ties. Besides, p value < 0.05 was considered statistically significant Ranjbar et al., 2019). Figure 2 shows the gel electrophoresis of 16SrRNA gene amplification in this PCR reaction.     Figure 3 shows the distribution of multidrug-resistant (MDR) P. aeruginosa strains isolated from raw meat and surface swab samples.

Determination of multidrug-resistant isolates
According to obtained data, all P. aeruginosa isolates harboured complete resistance to at least one antibiotic agent (100%). The prevalence of resistance of P. aeruginosa isolates of raw meat samples against at least 3 and more than six antibiotic agents was 69.56 and 17.39%, respectively. Prevalence of resistance of P. aeruginosa isolates of carcass surface swab samples against at least three and more than six antibiotic agents were 76 and 25%, respectively.

DISCUSSION
Debating the origin of bacteria in food is very challenging. In the present study, samples of raw meat and swabs were taken from the surface carcasses of animal species that were contaminated with P.
aeruginosa strains. In this regard, 6.57% of raw meat and 12% of carcass surface swab samples were contaminated with P. aeruginosa strains with a higher contamination rate for samples taken from cattle species.  P. aeruginosa in samples taken from cattle. Scarce researches are available in this regard. In a survey by Sheir et al. (2020), 4.00% of meat product samples were contaminated with P. aeruginosa owing to the needless handling and manipulation of meat samples and poor hygienic quality of raw materials. A higher prevalence of P. aeruginosa in cattle meat samples was reported by Benie et al. (2017). They found that 53.04% of cattle meat was contaminated by P. aeruginosa. Similarly, Benie et al. (2016) reported that 97.90% of beef samples were positive for P. aeruginosa strains. Other surveys were conducted on chicken meat (Mahato et al., 2020), camel meat (Osman et al., 2019), cattle meat (Odoi et al., 2021), frozen meat (Ibrahim et al., 2016) and meat products (Sofy et al., 2017) samples showed that the P. aeruginosa prevalence had a range between 3.00 to 80.00%. High adaptation of P. aeruginosa strain in different temperatures (4-42 • C) and water activities (72-97%) (Gu et al., 2016) may be another reason for its high prevalence among the examined samples. stock. Therefore, it is logical that the prevalence of antibiotic resistance in strains isolated from the carcass surface is higher than those of meat samples. In a survey conducted in China (Meng et al., 2020), Australia (Khan et al., 2020), Germany (Yayan et al., 2015) and Saudi Arabia (Khan & Faiz, 2016), P. aeruginosa strains harboured a high resistance rate toward penicillin, ampicillin, tetracycline, gentamicin and trimethoprim antimicrobial agents. Benie et al. (2017)  In conclusion, P. aeruginosa strains were detected in 8.54% of raw meat and carcass surface swab samples. The considerable prevalence of P. aeruginosa strains was accompanied by the high rate of bacterial resistance toward commonly used antibiotic agents, particularly penicillin, ampicillin, tetracycline, gentamicin and trimethoprim. The findings may show the high antibiotic resistance of P. aeruginosa and the potential role of raw meat and carcass surface swab samples in its transmission to the human population. Some strains harboured different antibiotic resistance genes, particularly blaCTX-M, blaDHA and blaTEM, and virulence factors, especially ExoS, algD, lasA, plcH and exoU.
These findings may show the role of raw meat and carcass surface swab samples as a source of virulence factors and antibiotic resistance genes.
It seems that the consumption of contaminated meat with virulent and resistant P. aeruginosa may cause severe foodborne diseases that resist antibiotic therapy. However, the role of contaminated meat as a hazard of foodborne infection has not been determined yet. Thus, several studies should perform to assess the role of meat and animal carcasses in the transmission of virulent and resistant P. aeruginosa foodborne diseases.
Samples collected from carcass surface swab samples harboured a higher prevalence rate, antibiotic resistance rate and distribution of virulence factors. This may show the higher contamination rate of animal carcass surfaces with resistant and virulent bacteria. As samples were collected at the end of the slaughter procedure (after washing), it seems that the washing of animal carcasses was not done properly. As isolates of carcass surface swab samples harboured higher antibiotic resistance, it may conclude that some isolates may transfer after carcass manipulation by meat inspectors and staff of the slaughterhouses.
The present study was limited to the low diversity of collected samples, especially the lack of samples collected from ovine, camel and buffalo species. The absence of molecular typing of P. aeruginosa isolates was another important limitation of this survey. An adequate number of collected samples, phenotypically and genotypically determination of antibiotic resistance patterns of P. aeruginosa isolates, and finally an examination of the distribution of MDR isolates were the most important strong points of this survey.